Cytochrome c release from mitochondria proceeds by a two-step process

Abstract

Cytochrome c is often released from mitochondria during the early stages of apoptosis, although the precise mechanisms regulating this event remain unclear. In this study, with isolated liver mitochondria, we demonstrate that cytochrome c release requires a two-step process. Because cytochrome c is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin, this interaction must first be disrupted to generate a soluble pool of this protein. Specifically, solubilization of cytochrome c involves a breaching of the electrostatic and/or hydrophobic affiliations that this protein usually maintains with cardiolipin. Once cytochrome c is solubilized, permeabilization of the outer mitochondrial membrane by Bax is sufficient to allow the extrusion of this protein into the extramitochondrial environment. Neither disrupting the interaction of cytochrome c with cardiolipin, nor permeabilizing the outer membrane with Bax, alone, is sufficient to trigger this protein's release. This mechanism also extends to conditions of mitochondrial permeability transition insofar as cytochrome c release is significantly depressed when the electrostatic interaction between cytochrome c and cardiolipin remains intact. Our results indicate that the release of cytochrome c involves a distinct two-step process that is undermined when either step is compromised.

Figure 1

Alterations in the electrostatic interaction between cytochrome c and cardiolipin influences Bax-induced cytochrome c release from isolated mitochondria. (a and b) Mitochondria (1 mg/ml protein) were incubated under strict non-MPT (described in text) conditions in either 150 mM KCl (trace 1), 100 mM KCl/sucrose (trace 2), or MSH (trace 3) buffer as described under Materials and Methods, and Bax (10 μg/ml) was added after a 5-min stabilization period. (a) Mitochondrial swelling was monitored continuously at 540 nm. A typical swelling curve induced by 150 nmol of Ca²⁺ per mg of protein and 5 mM P_i is shown as a positive control (trace 4). (b) Alternatively, with use of a TPP⁺-sensitive electrode, changes in Δψ of mitochondria incubated in the three different buffers were evaluated, after the addition of Bax. (c) Eight minutes after the addition of Bax, samples were centrifuged, and the resulting supernatants separated by SDS/PAGE and Western-blotted as described in Materials and Methods.

Figure 2

Oxidative stress mobilizes a unique pool of cytochrome c that involves peroxidation of cardiolipin. (a–c) Mitochondria (1 mg/ml) were incubated as described in Materials and Methods and treated with different ROS-generating systems. After a 5-min stabilization period, Bax and inducers of ROS were added. After 8 min, samples were centrifuged, and the supernatants were separated by SDS/PAGE and Western-blotted for the presence of cytochrome c as described in Materials and Methods. The concentrations of the different reagents are as follows: cumene hydroperoxide (250 μM), butylated hydroxytoluene (5 μM), Fe(II)SO₄ (60 μM)/ascorbate (500 μM), antimycin A (5 μg/ml) + ATP (1 mM). (d) Changes in mitochondrial (0.5 mg/ml) volume were evaluated in the presence of the different ROS-generating systems as follows: trace 1, cumene hydroperoxide; trace 2, Fe²⁺/Asc; trace 3, antimycin A; trace 4, shows a positive control induced by 150 nmol of Ca²⁺ per mg of protein and 5 mM P_i. (e) Duplicate samples (a–c) were separated by SDS/PAGE and Western-blotted for the presence of AK-2. (f) Mitochondria were incubated as described for a–c, and lipids were extracted after 13 min as presented in Materials and Methods. An aliquot was subjected to HPLC analysis, and quantitative analysis of the cardiolipin hydroperoxide content obtained from three independent experiments is presented. Data are expressed as means ± SD. *, significantly different from control mitochondria. BHT, butylated hydroxytoluene; CuOOH, cumene hydroperoxide.

Figure 3

Fe²⁺/Asc accentuates cytochrome c release stimulated by MPT in intermediate and high ionic strength buffers. (a) Mitochondria (0.5 mg/ml) were incubated as described in Materials and Methods, and swelling was monitored continuously at 540 nm. MPT was induced by 150 nmol of Ca²⁺ per mg of protein and 5 mM P_i in the presence or absence of Fe(II)SO₄ (60 μM)/ascorbate (500 μM). Trace 1, control; trace 2, control + Fe²⁺/Asc; trace 3, MPT; trace 4, MPT in the presence of Fe²⁺/Asc. (b) Mitochondria (1 mg/ml) were incubated as described in Materials and Methods. After stabilization, MPT was induced, and Ca²⁺ release into the incubation medium was monitored. Trace 1, control; trace 2, control + Fe²⁺/Asc, trace 3, MPT; trace 4, MPT in the presence of Fe²⁺/Asc. (c) Mitochondria (1 mg/ml) were incubated as described in Materials and Methods and, in some cases, stimulated to undergo MPT ± Fe²⁺/Asc. Samples were taken for Western blot analysis exactly 8 min after t₀ (a) to compensate for the shorter onset of MPT in the presence of Fe²⁺/Asc.

Figure 4

Model for the two-step release of cytochrome c from mitochondria.